Penetrating keratoplasty is widely used to restore vision in a number of corneal illnesses. In severe corneal dystrophy, inflammation or degenerative processes, penetrating keratoplasty is the only effective therapy to obtain visual rehabilitation. A major issue in this therapy is the method used to preserve a viable corneal tissue after its removal from the donor.
The cornea is an avascular tissue with a well defined organization, 1 mm thick peripherally and 0.5 mm thick centrally. The part exposed to the external environment is covered by a stratified, nonkeratinized epithelium formed by 3 to 4 layers of flattened squamous cells, 1 to 3 layers of midepithelial cells and a single layer of columnar basal cells attached to the basement membrane and the underlying stroma by an adhesion complex. During corneal storage, epithelium may be lost but if the basement membrane is undamaged, re-epithelialization after the transplant is usually rapid. The stroma is arranged in three distinct layers of extracellular matrix. Starting from the epithelium, these include a thin Bowman's layer, a middle lamellar stroma and a basement membrane (Descemet's membrane) that is generated by the endothelial cells lining the side of the tissue facing the aqueous humor. The other parts of the stroma are produced and maintained by the stromal fibroblasts, flat cells commonly termed keratocytes.
Because of the presence of salts, collagen and proteoglycans, the stroma is hypertonic with respect to both tears and aqueous humor. Water is less concentrated in the epithelial side likely because of drying through the epithelium layers. Similarly, glucose is less concentrated in the epithelial side because this metabolite flows from the aqueous humor to be largely utilized by the epithelium. The stroma contains proteoglycans (dermatan and keratan sulfate) with the former more concentrated in the epithelial side and the latter in the endothelial side. The corneal endothelium is a single layered, cuboidal endothelium forming a hexagonal mosaic lying on Descemet's membrane when viewed from the anterior chamber. The hexagonal cells are linked together by tight junctions which, however, do not form a complete seal around the cells. Rather, they are concentrated in the cell apical membrane. Junction integrity depends on the presence of Ca2+ in the surrounding medium. This organization allows the aqueous humor and its solutes to have access to the paracellular space. Under normal conditions, fluid influx is not followed by the swelling of cornea because an equivalent volume of fluid is actively removed by the pumping complex of the endothelium. The Na+-K+ ATPase is an essential part of this pumping system which therefore requires the ATP produced by the metabolic activity of endothelial cells.
To fully evaluate the function of endothelial cells, it is of interest to observe that these cells have, on the side facing the aqueous humor, the immunoglobulin family member, ICAM-1 (intercellular adhesion molecule-1), which serves as a coreceptor for the integrin LFA-1 (α L, β2), located in the leukocyte surface. ICAM-1 may also function as a receptor for hyaluronic acid (McCourt, P. A. G. et at. (1994) J. Biol. Chem. 269, 30081-30084). This indicates that endothelial cells interact with leukocytes when they reach the aqueous humor. It may also indicate that under normal conditions and in the absence of leukocytes, hyaluronic acid associates with this receptor to preserve the integrity of the endothelial layer.
Swelling of corneas: When the pumping function of the endothelium is lost, hypertonicity of stroma causes the corneal tissue to swell. Swelling results in the increase of corneal thickness and a decrease in clarity. Furthermore, there is a loss of proteoglycans from the stroma to the surrounding medium. Loss of endothelial function is the consequence of pathological events (e.g. dystrophies, degenerations, glaucoma). Aging may favor endothelial decompensation since the number of endothelial cells declines with age (about 50% from birth to old age). Since endothelial cells have limited regenerative capacity, damage to the endothelium integrity can only be compensated by the enlargement of residual cells which become thinner. Endothelial damage is also the major risk in the storage of corneas before transplantation. This event results in corneal swelling with loss of clarity and progressive endothelial cell death. Preservation of an intact endothelial layer is, indeed, a major goal in devising methods to preserve corneas before penetrating keratoplasty.
Storage of corneas: Whole ocular globes from donors can be stored in a moist chamber at 4° C., but should be used within 24 h. Preservation of corneas in the frozen state has been successfully exploited (Kaufman, H. E. and Capella J. A. (1968) J. Cryosurg. 1, 125-129). Before freezing, corneas are treated with increasing concentrations of DMSO and sucrose to prevent formation of intracellular ice. Difficulties in handling and transport of frozen corneas at a constant low temperature prevent the diffusion of this technique. Alternatively, corneas can be stored at 37° C. in organ culture medium (Doughman D. J. et al. (1976) Trans. Am. Acad. Ophtalmol. Otolaringol. 81, 778-793). Since culture media are supplemented with serum for optimal cell preservation, this method has the disadvantage of exposing the recipient eye to a residual amount of serum transported by the cornea at the moment of transplant. Animal serum may elicit an immune response while human serum may transmit viral diseases.
At present, the most convenient method for corneal preservation appears to be short-term storage in serum-free media at 4° C. At this temperature, the metabolic activity of endothelial cells is minimal. Thus, pumping function is lost.
Cornea swelling may be prevented by the addition of water-retentive compounds to the preservation medium. Among these, one of the most used is the deturgescent compound, dextran, either alone (McKarey, B. B. and Kaufman, H. E. (1974) Invest. Ophthalmol. Vis. Sci. 13, 165) or in association with the glucosaminoglycan chondroitin sulfate (Kaufman H. E. et al. (1991) Arch. Ophthalmol. 109, 864-868). However, chondroitin sulfate is a heterogeneous compound because of the varied distribution of the sulfate molecules within the polymer (Scott, 1995). As a result, the compositions to be used for corneal storage may vary between lots. In addition, due to the sulfate molecules, chondroitin sulfate carries a strong negative charge.
It has recently been suggested that this strong negative charge is detrimental to corneal preservation because the strong negative charges decrease the adhesion capability of corneal endothelium (Chen et al. 1996). Further, it has been reported that chondroitin sulfate can penetrate the cornea and favor its swelling, particularly upon rewarming the tissue from 4° C. to room temperature, before transplant (Kaufman et al. 1991). In an attempt to decrease the corneal swelling induced by chondroitin sulfate, cornea preservation compositions were formulated which contain deturgescent agents such as dextran, in combination with chondroitin sulfate (EP 0 517 972). However, dextran may penetrate the stroma during storage and may increase the swelling pressure on rewarming. In addition, it is now also clear that dextran can be toxic to the cornea, inducing senescence and degeneration (Chen et al. 1996).
Ogino et al. (Mokugan Zen-shi, Effect of a Newly Developed Corneal Storage Medium on Corneal Endothelium —Morphological Study by Scanning Electron Microscopy, 1995) describe a storage medium containing hyaluronic acid (HA). But the hyaluronic acid utilized by Ogino et al. had a molecular weight of 800,000. Such a high molecular weight HA is too viscous to be suitable as a storage medium component and must be used in combination with some other water-retaining component to prevent cornea swelling without augmenting the viscosity of the solution.
It is, therefore, an object of the present invention to provide a cornea storage fluid capable of providing suitable storage conditions for viable cornea, while avoiding the drawbacks of prior fluids.